CN100544685C

CN100544685C - The biological tissue treatment facility

Info

Publication number: CN100544685C
Application number: CNB2004800131981A
Authority: CN
Inventors: 梅津光生; 岩崎清隆; 尾崎重之; 守本祐司; 远藤修
Original assignee: Aubex Corp; Waseda University
Current assignee: Core tissue bioengineering Ltd; Waseda University
Priority date: 2003-05-15
Filing date: 2004-05-12
Publication date: 2009-09-30
Anticipated expiration: 2024-05-12
Also published as: EP1625832A1; CN1787792A; JP4444918B2; AU2004238128A1; AU2004238128B2; US8043854B2; WO2004100831A1; EP1625832B1; EP1625832A4; JPWO2004100831A1; US20060212074A1

Abstract

Put into transplanting of human body in the preparation of taking out such as valvular biological tissue from the animal health, the flow regime that is used in the cells exclude solution of getting rid of the germinal cell in the biological tissue equals the flow regime of graft receptor live body substantially, and described biological tissue is put into described liquid stream, so that described biological tissue effectively is immersed in the cells exclude solution.When soaking, preferably described biological tissue is put into cells exclude solution, rotate, use microwave radiation simultaneously.As a result, germinal cell evenly and reliably can be got rid of from biological tissue, so that can improve the bio-compatibility of biological tissue after transplanting.

Description

The biological tissue treatment facility

Technical field

The present invention relates to method of removing cell, more particularly, relate to the method for removing cell that when transplanting biological tissue, can strengthen the effect of eliminating cell from biological tissue.

Background technology

When the cardiac valve cisco unity malfunction of human body, can cause the difficulty that refluxes such as cardiac valve opening constriction and blood, described cardiac valve must be changed with replacement valve.As the replacement valve of present use, can use the mechanical valve prosthesis of making by artificial material, xenograft that takes out in the animal body and the allogeneic biovalve contributed by other people etc. such as pig.There is following problem in such valve.Although mechanical valve prosthesis is durable, receptor must be at his anticoagulant of use in life.On the other hand, in xenograft, receptor there is no need to use all one's life anticoagulant; The but long-time problems such as calcium deposition that take place have caused the dysfunction of valve, consequently must change the valve of damage with about 15 years interval with new replacement valve.In addition, for the allogeneic biovalve, because the limited amount of donor is not easy to obtain a large amount of allogeneic biovalves.

Wherein, xenograft has application prospect, because such valve of sufficient amount can be provided, and the patient there is no need to use throughout one's life anticoagulant after transplanting.Therefore, estimate that xenograft is more useful than other replacement valve, only need overcome the relatively poor defective of ruggedness and get final product.

In this article, known following method can suppress to transplant the back immunologic rejection, and improves the ruggedness (for example, referring to Japanese Patent No. 6-261933) of the xenograft that takes out in the animal body such as pig.In the method, at first xenograft is immersed in the cells exclude solution, in cholic acid or surfactant, so that eliminating is from the cell of described animal, as endotheliocyte and fibroblast (method of removing cell).The xenograft that to get rid of the germinal cell of its inside then already is immersed in and contains autogenous cell, as from the receptor human body (as described in the xenogenesis life state xenograft will be transplanted to as described in the human body) endotheliocyte and fibroblastic solution that contains cell so that described autogenous cell is seeded in (cell inoculation method) on the described xenograft.

But, in above-mentioned processing method, because the xenograft that takes out in animal body can not be handled described cells exclude process and cell inoculation process effectively, resulting xenograft can not obtain enough bio-compatibilities.In other words, because germinal cell remains in the described cells exclude process more or less, the bio-compatibility of resulting xenograft is owing to the existence of germinal cell reduces.

Under above-mentioned occasion, the inventor had carried out deep experimentation already, its objective is to overcome the problems referred to above.They have designed a kind of method that described cells exclude solution is provided, and are used in fact to be equivalent to the xenograft that the mobile flow velocity of blood of human body soaks xenograft and/or is immersed in described cells exclude solution with microwave radiation.As a result, they find significantly to have reduced by this method the quantity of residual germinal cell.

Summary of the invention

The present invention is based on above-mentioned discovery design.An object of the present invention is to provide effective eliminating and be present in method, so that after transplanting, improve the bio-compatibility of biological tissue such as the germinal cell in the biological tissue of xenograft.

In order to achieve the above object, the present invention relates to be used for when transplanting biological tissue, getting rid of the method for removing cell of germinal cell from predetermined biological tissue, comprise described biological tissue is immersed in the predetermined cells exclude solution, wherein, described cells exclude solution can flow with the state of the stream of beating, this is to realize by described cells exclude solution being applied predetermined pulse, and, biological tissue is put into the described stream of beating.By this method, the quantity of residual germinal cell is compared with conventional method and can significantly be reduced in the described biological tissue, thereby has improved the bio-compatibility of described biological tissue after transplanting.

In addition, the present invention relates to be used for when transplanting biological tissue, getting rid of the method for removing cell of germinal cell, comprise biological tissue is immersed in the cells exclude solution from predetermined biological tissue,

Wherein, put into the biological tissue of described cells exclude solution with microwave radiation.Above-mentioned purpose can also realize by this method.

Described (back a kind of) method can with get rid of by pair cell that solution applies predetermined pulse so that cells exclude solution flows with the state of the stream of beating, and biological tissue is put into described method of beating stream carries out simultaneously.Can further improve the effect of getting rid of cell by this method, and germinal cell almost completely can be got rid of from described biological tissue.

Microwave applies when rotate at described microwave radiation position at biological tissue.Like this, in fact can apply microwave to biological tissue equably along the rotation round direction, consequently, in fact can be with cell equably the nearly all position along the rotation round direction from biological tissue remove.

Brief description of drawings

Fig. 1 is a sketch map of indicating to use the schematic structure of biological tissue treatment facility in the present invention;

Fig. 2 is vertical cross-sectional schematic of driving pump;

Fig. 3 is the schematic elevational view that constitutes the holding device of described biological tissue treatment facility;

Fig. 4 is the decomposition diagram of the lid of described holding device;

Fig. 5 is the schematic cross sectional views of described holding device;

Fig. 6 is the right side view sub-anatomy of structure shown in Figure 5;

Fig. 7 is the schematic, exploded perspective view that the passage of inflow entrance forms parts;

Fig. 8 is the left side view sub-anatomy of structure shown in Figure 5;

Fig. 9 is the schematic, exploded perspective view that the passage of flow export forms parts;

Figure 10 (A) is the sketch map from a structure part shown in Figure 3, comprises described holding device;

Figure 10 (B) is that identical structure shown in Figure 10 (A) is in the state around state Rotate 180 shown in relative Figure 10 of back shaft (A) °;

Figure 11 is illustrated in the example 1,2 form of residual germinal cell quantity on the unit are, compares with comparative example 1;

Figure 12 is the microphotograph of the amplification of the biological tissue of acquisition in example 3; With

Figure 13 is the microphotograph of the amplification of the biological tissue of acquisition in example 4.

Implement best mode of the present invention

Now, will describe embodiment of the present invention in conjunction with the accompanying drawings.

Fig. 1 is the sketch map of the schematic structure of expression biological tissue treatment facility of the present invention.Shown in the accompanying drawings biological tissue treatment facility 10 is used to the cells exclude process and the cell inoculation process of the biological tissue of xenograft.The cells exclude process is to carry out before the xenograft that takes out in the animal body such as pig is transplanted to the human body.The cells exclude process is by xenograft being immersed in the cells exclude solution such as cholic acid, and zooblast (below be referred to as " germinal cell ") carries out so that get rid of, so that only obtain the substrate be made up of collagen etc.On the other hand, the cell inoculation process is to be immersed in the solution that contains cell of the cell that contains human receptor's (described xenograft will be transplanted to this human body in) (below be referred to as " autogenous cell "), so that autogenous cell is attached on the described substrate by the xenograft that will remove germinal cell wherein already.

Biological tissue treatment facility 10 comprises circulating device 10A, and the solution that is used to make cells exclude solution and contain cell passes through predetermined circuit cycle, and is connected the holding device 10B on the circulating device 10A, is used to keep xenograft.Notice that if unspecial explanation, cells exclude solution is collectively referred to as " solution " with the solution that contains cell.

Circulating device 10A comprises known air suction/feedway 11, driving pump 12 that form by polyurethane and that be connected with air suction/feedway 11, and the circulation canal of installing like this 13, make the solution of from driving pump 12, getting rid of return driving pump 12.

Air suction/feedway 11 has known structure, can be with air sucking-off from driving pump 12, and deliver air to driving pump 12.This paper has omitted the detailed description to described structure.

Driving pump 12 is pulsating pumps, can spray the stream of beating by produce spiral vortex in pump.More particularly, driving pump 12 has adopted the structure (Japanese patent application publication No. 2002-167836) that had been proposed by the applicant already shown in figure 2.More particularly, driving pump 12 comprises the superstructure 17 of hollow, in fact has tapered profile, it has inflow entrance 15 and flow export 16, and in fact substructure 18 of hollow has the cheese profile, it is positioned at below the superstructure 17, and flexible membrane 20, it will be separated into two parts by the space that structure 17 and 18 forms, i.e. internal space S 1 and S2.At the inflow entrance 15 shown in the right side of Fig. 2 is to form like this, so that be screwed into by the periphery wall of superstructure 17, and flow export 16 is arranged on the top of superstructure 17, referring to the upper end of Fig. 2.

Substructure 18 has the steam vent 22 that is communicated with air suction/feedway 11.At preset time, compressed air alternately is transported to lower inner space S 2 and from this space, gets rid of.When compressed air input and eliminating internal space S 2, diaphragm 20 is subjected to displacement, and increases or reduce the volume of upper interior space S 1.As a result, the solution from flow export 16 outputs forms the stream of beating.At this moment, in upper space S1, produced spiral vortex, shown as the dotted line among Fig. 2.In this state, be not easy to occur the quiescent centre.In an embodiment of the present invention, the pressure (malleation) that is transported to the air in the internal space S 2 is set to about 140mmHg-260mmHg, and internally in the space S 2 pressure (negative pressure) of the air of sucking-off be set to about-30mmHg is to-50mmHg, but, above-mentioned scope is not limited to these values.

Referring to Fig. 1, circulation canal 13 is that the closed-loop path by following form forms, and described solution flows out and flows into inflow entrance 15 and do not contact outside air from the flow export 16 of driving pump 12.More particularly, fluid passage 13 comprises that resistance forms device 23, and it produces flow resistance to the solution of output from flow export 16; The upstream tube 24 that is connected with flow export 16; Play the control valve 25 of strength control device effect, it is connected with the downstream of upstream tube 24; The connection tube 26 that is connected with the downstream of control valve 25; Be installed in the downstream of connection tube 26 and the downstream tube 27 that is connected with the inflow entrance 15 of driving pump 12; And the connection pump 28 that between connection tube 26 and downstream tube 27, provides.Notice that parts 24-28 connects by known joint 29.Below, for convenience's sake, the part that resistance forms device 23 upstreams that is positioned at of circulation canal 13 is known as " upstream tube L1 ", is known as " downstream tube L2 " and be positioned at the part that resistance forms device 23 downstreams.

Suppose it is under the situation of the Peripheral resistance of human body, resistance forms device 23 and is arranged on the centre of connection tube 26 separately, and is provided with the clamping part (not shown), is used for fixing connection tube 26.Because connection tube 26 is fixed by resistance formation device 23, if driving pump 12 pulses, the minimum blood pressure of upstream tube L1 will can not reach 0mmHg.Like this, simulated the artery blood flow of human body.Can be by the fastening degree of control connection pipe 26, the average pulse pressure of upstream tube L1 is adjusted to predetermined value.In the present embodiment, average pulse pressure is set at about 100mmHg, in fact it be equivalent to the average pulse pressure of human body.Notice that form device 23 as resistance, any parts that comprise variable orifice except clamping part can use, as long as it has and identical effect mentioned above.

Upstream tube 24, connection tube 26 and downstream tube 27 are to be made by vinyl chloride, but are not confined to this especially.As hereinafter disclosed, holding device 10B is arranged on the centre of upstream tube 24.Because xenograft is placed among the holding device 10B, and check-valves 30 is arranged in the downstream tube 27, described solution can circulate under the condition that does not reflux along direction shown in the arrow among Fig. 1.

Control valve 25 is installed in the upstream that resistance forms device 23, and plays a part controller, the intensity of the pulse pressure of control upstream tube L1.More particularly, control valve 25 is made by flexible material, as segment-like polyurethane or silicone, and can be by changing the pulse pressure intensity of pipe thickness control upstream tube L1.Notice that in the present embodiment, the pulse pressure intensity of upstream tube L1 is set to, for example, 100mmHg (average pulse pressure) ± 20mmHg is so that near the human body pulse pressure.

Connect pump 28 and be the stream of beating and form pump with structure identical with driving pump 12.Use similar Reference numeral to represent and driving pump 12 corresponding similarly structure members, and omit all further instructions.Notice that connecting pump 28 is to install along such direction, make described solution flow into, and flow out by flow export 16 by inflow entrance 15.The steam vent 22 that connects pump 28 leads to the external world.Diaphragm 20 is according to the flow of solution displacement.The amount of the solution among the circulating device of packing into the 10A allows useful load a little less than the maximum of device 10A, and the displacement that connects the diaphragm 20 of pump 28 is slightly less than greatest physical and allows displacement.Therefore, connect pump 28 inhibitory action is provided.More particularly, when solution during, diaphragm 20 displacements, thereby bring high blood pressure down by described connection pump.Therefore, connect pump 28 and be installed in the downstream that resistance forms device 23, and play a part the pulse pressure reducer, be used to weaken the fluid pressure of the solution of downstream tube L2.In the present embodiment, the pressure of the solution by connecting pump 28 is set to about 10mmHg, so that suitable with the pressure of human body left atrium.

Note, connect pump 28 and can be installed between control valve 25 and the connection tube 26.

The solution that flows through circulating device 10A is introduced into the xenograft that holding device 10B wherein will be immersed in the solution to be kept.In the present embodiment, the xenograft as carrying out cells exclude process and cell inoculation process has used porcine aortic valve.Holding device 10B is installed in the position that having of circulation canal 13 approached the blood flow state of human aorta lobe part.Say that simply holding device 10B is connected with the middle of upstream tube 24.Below, for convenience of explanation for the purpose of, the part that is positioned at holding device 10B upstream of upstream tube 24 is known as inlet tube 24A, and the part that is positioned at holding device 10B downstream is known as outlet 24B.Notice that holding device 10B can freely be connected with any position of circulation canal 13, by the position of suitable selection circulation canal 13, its state (as pressure) approaches the state by the blood flow of target biological tissue.

Referring to Fig. 3, holding device 10B comprises tubular base 33, and it can be placed on the predetermined surperficial F, is positioned at the main body 35 of base 33 tops and is positioned at the

support component

36,36 of the left and right sides of base 33, is used for supportive body 35.

Main body 35 comprises box-shaped lid 38, sees that from the front it has hexagonal shape; The holding device 39 that is used for fixing xenograft V, it is arranged in the lid 38; Have octagon (from the front), be used to support holding device 39 and be the member of installing along the inwall of lid 38 40; Irradiator 42 (radiation appliance), it is positioned at lid 38 back, is used for the xenograft V that remains on holding device 39 is applied microwave;

Back shaft

44,44 is respectively from the right side of member 40 with pass on left lid 38 and extend to

support component

36,36; And two pipes, i.e. ingress pipe 46 that links to each other with holding device 39 and delivery pipe 47.

Referring to Fig. 4, lid 38 has box structure, and it is made of two parts of longitudinal subdivision, more particularly, comprises the bonnet 52 that is positioned at positive protecgulum 51 and is positioned at the back side.The butted part of protecgulum 51 and bonnet 52 is to form in the position of intersecting with back shaft 44,44.In the outer ledge part of described butted part, in fact, formed flange plane 54,54 along described outer ledge part.On flange plane 54,54, a plurality of through hole 55 (not shown) that are used for screw (volts) have been formed.When flange plane 54,54 contacts with each other Face to face, and when connecting with screw, protecgulum 51 and bonnet 52 are integrally combined.When described screw removes from described integrated morphology, protecgulum 51 can separate, and separates with bonnet 52.In addition, microwave radiation mouth 57 has the rectangular shape of opening forward, and it is to form around the central authorities of bonnet 52 inside.Radiation port 57 is to form like this, so that in fact forward corresponding to the xenograft V (referring to Fig. 3) that is kept by holding device 39.By this device, the microwave by irradiator 42 is carried backward by radiation port 57, and is applied to forward on the xenograft V that is positioned at holding device 39.In addition, protecgulum 51 and bonnet 52 have such structure, can prevent when holding device 39 applies microwave that from radiation port 57 microwave from revealing to the external world.Note, provide door-plate 59 at the front end of protecgulum 51.Door-plate 59 usefulness known materials are made, and can see the state of holding device 39 by it, and can prevent that by it microwave from revealing to the external world.

Referring to Fig. 5, holding device 39 comprises in the accompanying drawings the acrylic acid cylindrical part 61 that extends along dextrad and left-hand, be positioned at the inflow part 62 of the right end of cylindrical part 61, be positioned at the outflow part 63 of its left distal end, and be used for xenograft V is installed in the installing space 64 that flows between part 62 and the outflow part 63.In the holding device 39, described solution flows into cylindrical parts 61 from flowing into part 62, by installing space 64, and flows to the outside from flowing out part 63, as following disclosed.

Cylindrical part 61 all has opening at each end of bearing of trend.In described open end, be provided with and flow into part 62 and outflow part 63.Thin aluminum sheet 66 is sticked on the whole outer surface of cylindrical part 61, in fact be equivalent to except the central part of installing space 64.Adopt this structure, the microwave (referring to Fig. 3) that provides by irradiator 42 can only by in fact with the central inner peripheral portion transmission of installing space 64 corresponding cylindrical parts 61.As a result, microwave can focus on middle body; Meanwhile, can avoid improving the temperature of installing space 64 Outboard Sections.

Referring to Fig. 5 and 6, flow into part 62 and comprise end component 68, it engages with the outer surface of cylindrical part 61, and the passage formation parts 69 that are positioned at end component 68.

End component 68 is made into vessel form, as shown in Figure 6, has the hole in the left side of its bottom.More particularly, the sidewall 72 that end component 68 has the lower wall 71 that is positioned at the accompanying drawing right side and forms continuously in the circumferential edge of lower wall 71, and in fact extend vertically up to (towards the left side of Fig. 6) lower wall 71.Lower wall 71 has through hole 73 near core.Sidewall 72 has the screw thread 74 in the internal circumferential formation of opening one side.Screw thread 74 engages with the screw thread 75 that forms on the periphery of cylindrical part 61.

Referring to Fig. 5-7, passage forms parts 69 and comprises the conical pipe 77 that has with the hollow of cone analogous shape, and the insertion tube 78 that will insert conical pipe 77.

Conical pipe 77 comprises near installing space 64 localized cylindricality tops 79, skirt shape part 80 is 79 right sides that expand outwardly Fig. 6 gradually from the top, and begin from the centre in the accompanying drawings to extend along the direction on right and a left side is linear, the right end of the skirt shape part 80 of flanged edge part 81 in Fig. 6 forms continuously, and inner space 83 forms in part 79-81, and the right side in Fig. 6 and left side are in communication with the outside.Notice that explanation passage below forms the part of parts 69, if not otherwise specified, term " front edge side " expression conical pipe 77 is near a side at tops 79, and a keep to the side side of part 81 of term " trailing edge side " expression conical pipe 77.

On the outer surface at top 79, formed locking groove 85 along described circumference.When the end portion (as xenograft V) of the Sanguis sus domestica tubing B that comprises aortic valve is placed on the locking groove 85, and with the bonded block (not shown) fixedly the time,, can limits the B of vascular tissue 79 is taken out from the top referring to Fig. 5.

On skirt shape part 80, the external diameter of marginal portion 81 is set at the internal diameter that equals cylindrical part 61, so that marginal portion 81 just in time is bonded in the cylindrical part 61.

Marginal portion 81 is made into ring form.The external diameter of marginal portion 81 is set for the internal diameter of the sidewall 72 that equals end component 68.Adopt this structure, marginal portion 81 can just in time be combined in the end component 68.On the inner circumferential surface of marginal portion 81, formed screw thread 87.

Referring to Fig. 6, inner space 83 has the opening portion that is positioned at right end.Inner space 83 comprises bottom space 89, and the top of insertion tube 78 will be inserted wherein by opening portion.Main channel 91 is communicated with bottom space 89, and extends towards the opening portion 90 that forms at front edge side, and provides four lateral access 92 in the main channel around 91, is communicated with bottom space 89.Bottom space 89 has the inner space, and its shape is consistent with the top of insertion tube 78, can just in time be bonded on the insertion tube 78.Main channel 91 is made into the bellmouth form, its internal diameter from the top 79 the beginning inwardly dwindle, but, be not limited thereto.Referring to Fig. 7, on the surface of the inclination of skirt shape part 80, formed lateral access 92.More particularly, four lateral access 92 are spaced with what equate along the circumferencial direction of skirt shape part 80.Should be pointed out that in the present embodiment, the gross area of the opening portion of lateral access 92 is set at the area that equals main channel 91.

Referring to Fig. 6, insertion tube 78 is included in the spherical part 94 that front edge side has spherical shape, the cylindrical portion 95 that external diameter is littler than the external diameter of spherical part 94, and be formed into spherical part 94 continuously by step shape part.Outer cylindrical part 96 is bonded on around the outer surface of cylindrical portion 95, so that can rotate mutually, annular collar part 97 is arranged on the trailing edge side of outer cylindrical part 96 continuously, and its external diameter is less than the internal diameter of the through hole 73 of end component 68, and inner space 99 is formed in spherical part 94 and the cylindrical portion 95.Externally on the outer surface of cylindrical portion 96, formed screw thread 101.Screw thread 101 engages with the screw thread 87 that 81 the inside in the marginal portion forms.By this structure, insertion tube 78 can be connected with conical pipe 77.

Inner space 99 comprises foot passage 103, and solution can flow by it, and top-portion apertures 104 is communicated with foot passage 103, and in the front edge side opening of spherical part 94, and four side openings 105 are formed on around the top-portion apertures 104, and is communicated with foot passage 103.When the front edge side of insertion tube 78 was attached in the inner space 83 of conical pipe 77, top-portion apertures 104 stably was communicated with main channel 91.It is along the circumferential direction spaced apart with what equate on the surface of spherical part 94 that side opening 105 is actually.When insertion tube 78 is attached in the conical pipe 77, side opening 105 rotatably changes the position, from can be fully becoming the closed position that cuts off fully of getting in touch with all lateral access 92 with largest connected position change that all lateral access 92 are communicated with, this process is by managing 78 and 77 mutual rotation realization.Therefore, insertion tube 78 is by changing its Position Control flow, equals to flow out the position of the total flow of main channel 91 from the total flow of the solution that flows out all lateral access 92, and the total flow that changes to the solution that flows out all lateral access 92 is actually zero position.On this meaning, insertion tube 78 plays a part to be similar to variable throttle valve, controls the flow of effusive solution in the passage 92 from the side by it.

Referring to Fig. 5,8 and 9, the shape that flows out part 63 partly is different from the shape that flows into part 62; But, have identical substantially structure member, and work in fact similar mode.

Therefore, used similar Reference numeral flowing out on the part 63, so as expression with flow into the part 62 corresponding identical or equivalent configurations parts that flow out parts 63, and omission or simplified explanation to all technology.Only to remarking additionally with the difference that flows into part 62.

The end component 68 that flows out part 63 is made into the form of similar nut.In fact, the central authorities of parts 68 provide screwed hole 107 endways, so that extend along left and right directions in Fig. 8.

The conical pipe 77 that flows out part 63 is different with the shape that flows into part 62, and marginal portion 81 is formed into skirt shape part 80 continuously.Specifically, marginal portion 81 is made into the screw thread-like rod type, and its external diameter is less than the maximum outside diameter of skirt shape part 80.The outer peripheral portion of marginal portion 81 engages with the screwed hole 107 of end component 68.Conical pipe 77 has gathering sill 109, is used for inserting the heat sensor (not shown) here.Gathering sill 109 is arranged on 83 outsides, inner space.Groove 109 comprises the

first gathering sill

109A, and 81 trailing edge side extends to the surface of the inclination of skirt shape part 80 from the marginal portion for it, and the second gathering sill 109B, and it is at the central fascicle of the first gathering sill 109A, and extends to main channel 91.Notice that among gathering sill 109A and the 109B two or any can be saved.

The insertion tube 78 that flows out part 63 is different with inflow part 62 on its global shape.On insertion tube 78, cylindrical portion 95 is extended to front edge side, replaces spherical part 94.Top-portion apertures 104 is arranged on the front edge side of cylindrical portion 95.Four side openings 105 along the circumferential direction are arranged on the outer surface of the front edge side of cylindrical portion 95 with the interval that in fact equates.

Notice that at Fig. 5, the stop member in 6 and 8 is the O shape ring that is used to seal.

Referring to Fig. 3, member 40 is supporting holding device 39, so that rotate in its inside.More particularly, insertion tube 78,78 is in fact outstanding outward from the mediad of end component 68,68, and they are connected on the roll tube 112,112 by pipe nut 111,111.Roll tube 112,112 is connected on the part of member 40 separately by the bearing (not shown), and is projected into member 40 outsides.Holding device 39 is bonded in the member 40, so that roll tube 112,112 can extend with 45 ° angle as shown in Figure 3 obliquely.Holding device 39 can rotate (rotation) around roll tube 112,112.On the outer surface of member 40, and be positioned at the position of flowing out the outstanding side of part 63 (the upper right face of Fig. 3) near roll tube 112, provide have motor and gear etc. from rotary driving device 114.Holding device 39 rotates in member 40 from rotary driving device 114 by starting.In addition, roll tube 112,112 is connected with delivery pipe 47 with ingress pipe 46 respectively, connects by being fixed on the structural swivel joint of installing near member 40 115.As swivel joint 115, used known joint, it can connect two types cast parts (roll tube 112,112 and pipe 46,47), allows simultaneously to rotate mutually and interconnect.By described joint, even roll tube 112,112nd rotates from rotary driving device 114 by operation, revolving force can not be delivered on each pipe 46 and 47 yet.

Irradiator 42 is with the known devices of magnetron (not shown) as the microwave source.This paper has omitted the detailed description to this device.

Back shaft 44 is pipes of hollow, and the end portion of ingress pipe 46 and delivery pipe 47 is included.More particularly, formed keyhole 117 being positioned near the inner and position of

support component

36,36 of the outer surface of each back shaft 44,44.To insert keyholes 117 from the

pipe

46 and 47 of extension on the roll tube 112,112.

Support component 36 comprises first support section 120 that is installed in the inboard, be used for supported axle 44,44 rotatably and be installed in the connection supports part 122 in first support section, 120 outsides, be used to support swivel joint 121, the structure of this joint is identical with the structure of above-mentioned swivel joint 115.Delivery pipe 47 by support component 36 swivel joint 121 (left side among Fig. 3) that supports and the left side of inserting back shaft 44 rotatably is connected mutually with outlet 24B.On the other hand, the swivel joint 121 (right side among Fig. 3) that supports by support component 36 can be connected with inlet tube 24A mutually rotatably with the ingress pipe 46 that inserts back shaft 44 right sides.In addition, (right side among Fig. 3) provides the rotating driving device 124 with motor and gear etc. on support component 36.Can be by handling rotating driving device 124 rotation support shaft 44,44.As indicated above, when back shaft 44,44 rotated, member 40 and holding device 39 rotated (rotation) around back shaft 44,44 simultaneously.The ingress pipe 46 that inserts back shaft 44 and delivery pipe 47 be designed to separately during rotation not can with base 33 mutual interference mutually.More particularly, they have suitable length, and are installed into by the space between base 33 and the back shaft 44.Referring to Figure 10, when back shaft 44 during from state Rotate 180 shown in Figure 10 (A) ° (half cycle), member 40 is rotated.As a result, on the holding device 39/down and the position on a right side/left side concern and put upside down, referring to Figure 10 (B).More particularly, holding device 39 not only can rotate but also can pass through rotating driving device 124 by from rotary driving device 114 rotations.When holding device 39 rotation, on the B of vascular tissue/down and the position on a right side/left side concern and put upside down.Note the switch of the Reference numeral 125 expression rotating driving devices 124 among Fig. 3.Note, chiller can be provided, be used to cool off the periphery of ingress pipe 46, improve so that prevent solution temperature.

The process of xenograft V being put into the holding device 10B that makes as stated above will be described below, and the function of holding device 10B in setting-up time.

At first, under state shown in Figure 3, roll tube 112 discharges from swivel joint 115.Then, will manage nut 111 by rotation and unclamp, and from holding device 39, take out, so that holding device 39 is taken out from roll tube 112,112.Then, take out end component 68,68, and will flow into part 62 and outflow part 63 is taken out from cylindrical part 61.Then, at the top 79 that keeps flowing into part 62,79 and flow out the top 79 of part 63 can condition respect to one another under, referring to Fig. 5, top 79 is inserted at the two ends that will comprise the Sanguis sus domestica tubing B of aortic valve V respectively, 79, be separately fixed on the locking groove 85,85 by the bonded block (not shown) then.The B of vascular tissue is set to such direction, makes that solution can be by the right side flow direction left side of aortic valve V (guide valve) from Fig. 5.Then, the inflow part 62 that connects by the B of vascular tissue and flow out part 63 and return cylindrical part 61 once more, and end component 68,68 is connected with cylindrical part 61, and holding device 39 is bonded in the roll tube 112,112, referring to Fig. 3.Like this, just finished the assembling of the B of vascular tissue.

In the cells exclude process, cells exclude solution is transported to the insertion tube 78 that flows into part 62 from ingress pipe 46 by roll tube 112.At this moment, the lateral access 92 that the

insertion tube

78,78 that flows into part 62 and flow out part 63 is set at side opening 105 and conical pipe 77 is in the position of complete connected state, referring to Fig. 5.By this structure, flow to the cells exclude solution that flows into part 62 and organize B by the top-portion apertures 104 and main channel 91 intravasations that flow into part 62, by aortic valve V, and enter the main channel 91 of flowing out part 63.Simultaneously, the flow of solution of same amount is crossed side opening 105 and is flowed into the subchannel 92 of part 62, by the inner space of cylindrical part 61, and enters the lateral access 92 that flows out part 63, so that walk around the B of vascular tissue.Cells exclude solution is by the main channel 91 and the lateral access 92 of outflow part 63, and outflow from the insertion tube 78 that flows out part 63, is discharged in the delivery pipe 47, referring to Fig. 3 by roll tube 112.Therefore, constituted the first passage that is interconnected by tubulose biological tissue (B of vascular tissue) in the main channel 91 and the top-portion apertures 104 that flow into part 62 and flow out on the part 63, and constituted second fluid passage that is interconnected by circuitous route flowing into part 62 and the lateral access 92 and the side opening 105 that provide in the part 63 each are provided in the formation of the outside of biological tissue (B of vascular tissue).

When stating the cells exclude process on the implementation, operation irradiator 42 and from rotary driving device 114.Holding device 39 is rotations, and the B of vascular tissue in remaining on holding device 39 applies microwave simultaneously.By this operation, can on the whole circumference direction of the B of vascular tissue, implement the cells exclude operation equably.Described cells exclude operation preferably in checking the B of vascular tissue and carry out under the condition of outer solution temperature, is inserted the first and second gathering sill 109A by using, and the heat sensor (not shown) (referring to Fig. 5) of 109B is finished.Under this occasion, in fact cells exclude solution can flow the inside and outside of the B of vascular tissue with identical flow velocity, so that there is not the temperature difference between them.Can check the temperature difference between them by the heat sensor (not shown) that inserts the first and second gathering sill 109A and 109B.Notice that when inside and outside temperature reached human body temperature (for example, 37 ℃), irradiator 42 stopped microwave radiation automatically.Otherwise when temperature was lower than body temperature, it automatically began microwave radiation.It is 2.45GHz that the irradiator 42 of this embodiment can apply frequency, and power is the microwave of about 0-1200W.Notice that described temperature sensor can import first passage by being arranged on delivery pipe 47 intermediary holes.

On the other hand, in implementing the cell inoculation process, stopped applying the rotation of microwave and holding device 39 by irradiator 42.By rotating and unclamp collar portion 97, and cylindrical portion 95 is rotated to the state that the contact between side opening 105 (referring to Fig. 5 etc.) and the lateral access 92 cuts off fully from state shown in Figure 5 change the position of

insertion tube

78,78 in holding device 39.Then, the solution that will contain cell is transported to the insertion tube 78 that flows into part 62.The solution that contains cell arrives the B of vascular tissue by top-portion apertures 104 and the main channel 91 that flows into part 62 then, by aortic valve V, and the main channel 91 of inflow and outflow part 63, and from the insertion tube 78 that flows out part 63, get rid of subsequently.In this operating period,

insertion tube

78,78 is placed on the position that contact between the lateral access is cut off state.Therefore, the solution that contains cell can not flow to the B of vascular tissue outside, and is different with the situation of cells exclude process above-mentioned.

In implementing the cell inoculation process, started rotating driving device 124 as shown in Figure 3.Around back

shaft

44,44 rotations, consequently holding device 39 is rotated into topsy turvydom along direction shown in Figure 10 to holding device 39 with member 40.Because holding device 39 rotation, the autogenous cell that contains the solution of cell can be equably attached on the B of vascular tissue, and does not have gravitational effect.

Wait explanation by using the method for 10 eliminatings of biological tissue treatment facility and inoculating cell below in conjunction with Fig. 1.

At first, the B of vascular tissue that will comprise aortic valve V takes out in the animal body such as pig.As stated above the B of vascular tissue is put into holding device 10B.Cells exclude solution is injected biological tissue treatment facility 10, then circulation.As cells exclude solution, sodium lauryl sulphate (SDS), or the surfactant of Triton X-100 have been used such as deoxycholic acid (cholic acid).At this moment, cells exclude solution circulates in biological tissue treatment facility 10, keeps the state of described solution to approach the human body flow regime as stated above simultaneously,

When the push switch (not shown), suction/feedway 11 shown in Figure 1 is activated.Then, cells exclude solution circulates by circulation canal 13 by the pulsation of driving pump 12.More particularly, the cells exclude solution of ejection is crossed upstream tube L1 with the pressure current that in fact is equivalent to the common large artery trunks pressure of human body from driving pump 12, by being arranged on the intermediary holding device 10B of upstream tube L1, and arrive resistance and form device 23, so that produce the resistance that is equivalent to the human peripheral resistance.Form device 23 effusive cells exclude solution by connecting pump 28 from resistance, and flow into driving pump 12 with the pressure of about 10mmHg of the pressure that in fact is equivalent to left atrium.

At this moment, on holding device 10B, it is inside and outside that the cells exclude flow of solution is crossed the B of vascular tissue that remains in the holding device 39, and its flow regime is equivalent to flow through the state of the aortic blood flow of human body; Meanwhile, the B of vascular tissue that rotates by the rotation of holding device 39 is as stated above applied microwave.Like this, various types of germinal cells (endotheliocyte, fibroblast, smooth muscle cell) treating the preponderant disease instead of the secondary disease is got rid of in the B of vascular tissue of animal.As a result, stayed the substrate of only forming by collagen.In the present embodiment, it is 2.45GHz that microwave is set to frequency, and power is about 100W-500W, but is not limited to this condition.But, microwave power can change in preset range, as long as microwave can not produce any degeneration (harmful) to the B of vascular tissue, and produces predetermined cells exclude effect just.In addition, can different electromagnetic wave and the sound waves of frequency of utilization.Moreover, even the mobile not similar with blood flow of cells exclude solution also is feasible.In other words, beat stream also be acceptable.

After cells exclude solution is got rid of, normal saline is injected biological tissue treatment facility 10 from biological tissue treatment facility 10., by after the water washing of physiological cycle salt normal saline is got rid of from biological tissue treatment facility 10 at device 10.Then, will directly inject the first passage of holding device 39, so that close the opening that flows into part 62 and flow out part 63 such as the adhesive of fibronectin.Like this, the B of vascular tissue that had got rid of cell already and remain in the holding device 39 is soaked preset time in described adhesive.Then, adhesive is got rid of from holding device 39, and will be contained the solution injection holding device 39 of cell.The solution that contains cell prepares by the following method: gather the autogenous cell (endotheliocyte, fibroblast and/or smooth muscle cell) of receptor, cultivate preset time, and add in the predetermined culture fluid.As culture fluid, any culture fluid can use, as long as it can be used for the cell inoculation process.The example of this culture fluid comprises M199 (culture medium 199 is produced by LifeTechnologies company).Identical with the situation of adhesive, the B of vascular tissue that had got rid of cell is already being contained the solution soaking preset time of cell, start circulating device 10A then, so that make the solution that contains cell in 10 circulations of biological tissue treatment facility, the blood flow of its flow regime and human body is suitable.Like this, the B of vascular tissue that had got rid of cell is already put into being equivalent to and autogenous cell is inoculated among the B of vascular tissue by the mobile solution that contains cell of the aortic blood flow state of the mankind.Before producing the stream of beating, start rotating driving device 124 (referring to Fig. 3) by circulating device 10A.Like this, the B of vascular tissue that is immersed in the solution that contains cell rotates in the mode of putting upside down, so that will contain the actual whole zone that the solution of cell is combined in the B of vascular tissue equably, and does not have the influence of gravitational effect.Notice that this rotary operation can carry out continuously during generation contains the stream of beating of solution of cell.

According to a kind of like this embodiment, can significantly improve cells exclude effect and cell inoculation effect.Meanwhile, cells exclude process and cell inoculation process can be undertaken by certain operating sequence, simultaneously the B of vascular tissue are remained in the holding device 39.Can carry out simply at short notice with the processing of transplanting relevant xenograft.In addition, because this serial operation can be carried out in the passage of a sealing, xenograft can keep clean, and prevents from simultaneously to pollute.

Then, the inventor tests, so that checking is based on cells exclude effect of the present invention and cell inoculation effect.

(1) cells exclude effect experiment

[example 1]

In example 1,37 ℃ cholic acid are used as cells exclude solution.Cholic acid is injected circulating device 10A, and allow it flow, so that be equivalent to the blood flow (stream of beating) in the human large artery trunks.Allow the Sanguis sus domestica tubing that comprises aortic valve in described liquid stream, treat separately 24 hours.At this moment, circulating device 10A works under the following conditions: mean flow rate: 5L/ minute, the speed of beating of driving pump 12 (whipping speed): 70 times/minute, the average hydraulic pressure of cholic acid: about 90mmHg, and the maximum pressure of liquid and minimum pressure can be equivalent to the minimum and maximum pulse pressure of typical human body respectively.After handling, examine under a microscope vascular tissue, and by the camera enlarged image.The germinal cell (endotheliocyte and fibroblast) that is retained in the vascular tissue is counted, so that obtain unit are (1mm ²) average germinal cell quantity.

As a result, referring to Figure 11, about 850 germinal cells have been kept on the unit are.

[example 2]

In example 2, except the condition of example 1, vascular tissue is applied predetermined microwave, simultaneously by holding device 10B rotation.Used the microwave of frequency here, and under following three kinds of different conditions, applied: the power of 100W 8 hours, the power of the power of 500W 12 hours and 500W 24 hours by changing power and radiated time as 2.45GHz.Under all occasions, vascular tissue rotates 4 times with the mode per minute of rotation.In the mode identical the germinal cell that is retained in the vascular tissue is counted, and obtained unit are (1mm with example 1 ²) average cell quantity.

As a result, when the power with 100W applies 8 hours microwave, about 380 germinal cells on unit are, have been kept.On the other hand, apply at power under the condition of microwave of 12 hours and 24 hours, do not observe the germinal cell of reservation with 500W.In addition, in fact the albumen that is comprised in the original structure be excluded.Under all occasions, collagen and elastin laminin are in fact unaffected.

[comparative example 1]

Comparative example as example 1 and 2 has carried out an experiment.To be immersed in the cholic acid in example 1 employed vascular tissue, in container, leave standstill 24 hours.The germinal cell that keeps in the vascular tissue is counted, so that obtain unit are (1mm in the mode identical with example 1 ²) average germinal cell quantity.

As a result, referring to Figure 11, about 970 germinal cells have been kept on the unit are.

Under the initial conditions before carrying out the cells exclude process, be used for example 1 and 2 and the vascular tissue of comparative example 1 in unit are (1mm ²) 1880 germinal cells of existence.

According to above result, obviously, the quantity of the germinal cell of getting rid of in example 1 and example 2 is obviously greater than the quantity of the germinal cell of getting rid of in comparative example 1.Therefore, verified high cells exclude effect.Specifically, be immersed in occasion (example 1) in the stream of beating of cholic acid simply when the extra occasion of implementing microwave radiation is compared except immersion treatment in vascular tissue relatively, cells exclude better effects if under latter event.When microwave frequency during from 100W to 500W, germinal cell can not remain.Note, although omitted detailed description here, above the microwave mentioned can be applied in the vascular tissue that is immersed in the cholic acid, allow its to keep transfixion.The cells exclude effect of this situation is better than the effect of comparative example 1.

(2) experiment of cell inoculation effect

[example 3]

In example 3, after the cells exclude process, washed vascular tissue about 1 hour, and in fibronectin, soaked 4 hours with normal saline.Meanwhile, from the live body of the receptor that will transplant, take out autogenous cell (endotheliocyte), so that preparation contains the solution of cell.Here the employed solution that contains cell is to prepare by the following method: cultivated the autogenous cell gathered on the culture plate of M199 5 days containing, get rid of the autogenous cell of cultivating on the culture plate with trypsin, and add among the M199.Described cell is replenishing FBS (hyclone IWK-500 is produced by Iwaki), antibiotic (penicillin/streptomycin mixed solution), and cultivate among the M199 of FGF-2 (producing by Pepro Tech Ec Ltd.).

Then, vascular tissue was soaked in containing the solution of cell about 4 hours, rotate vascular tissue simultaneously up and down.After the cell inoculation process, examine under a microscope vascular tissue, and with the enlarged image of camera vascular tissue.Observation is seeded in the state of the autogenous cell (endotheliocyte) in the vascular tissue.

As a result, find that autogenous cell is inoculated in the in fact whole vascular tissue, referring to Figure 12.In fact, at the unit are (1mm of vascular tissue ²) on average combine about 850 autogenous cells.Unit are (1mm in vascular tissue ²) last ratio (granularity) average out to about 18% by the occupied area of autogenous cell.

[example 4]

In example 4, after with the step process vascular tissue identical, allow the solution that contains cell under the flox condition identical, in circulating device 10A, circulate with example 1 with example 3.

More particularly, vascular tissue be immersed in the solution that contains cell keep about 48 hours of transfixion after, it was rotated up and down about 4 hours.Then, allow the flow of solution that contains cell, so that be equivalent to the blood flow in the human body large artery trunks.Vascular tissue was stayed separately in the described liquid stream one hour.Then, use the mode identical to observe the state that is seeded in the autogenous cell in the vascular tissue with example 3.

As a result, find that in fact autogenous cell is seeded in all sites of vascular tissue equably, and autogenous cell is along the flow direction arrangement of the solution that contains cell, referring to Figure 13.In addition, compare with example 3, cell density has improved, and this is because the propagation of autogenous cell.In the present embodiment, be combined in unit are (1mm ²) vascular tissue on the quantity average out to about 2170 of autogenous cell.At unit are (1mm ²) vascular tissue on by ratio (density) average out to about 63% of the occupied area of autogenous cell.

[example 5]

Example 5 is to implement according to the mode identical with example 4, and different is, keeping vascular tissue actionless step and step of rotating vascular tissue up and down in containing the solution of cell is by in sequence opposite.More particularly, in the present embodiment, the vascular tissue that is immersed in the solution that contains cell rotated up and down about 4 hours, under static state vascular tissue was immersed in the solution that contains cell 48 hours then.Then, allow the described flow of solution that contains cell,, and allow vascular tissue stay separately in the described liquid stream one hour so that the blood of human body that is equivalent in the large artery trunks flows.Other conditions are identical with example 4.Then, observe the state that is seeded in the autogenous cell in the vascular tissue according to the method identical with example 4.

As a result, density is bred and increased to autogenous cell in the mode identical with example 4.In addition, compare with example 4, autogenous cell is seeded in all sites more equably.

[example 6]

Example 6 is to carry out in the mode identical with example 5, and different is, vascular tissue is stayed according to the time in the liquid stream of the solution that contains cell of the condition preparation identical with example 5 be set to 48 hours.

As a result, the in fact whole surface of the cell proliferation of inoculation and covering vascular tissue.

[example 7]

Example 7 is to carry out in the mode identical with example 6, and different is to have changed the flox condition of the solution that contains cell.More particularly, the mean flow rate that will contain the solution of cell is set at 2L/ minute, and the average fluid pressure that will contain the solution of cell is set at about 20mmHg.

As a result, in fact obtained the effect identical with example 6.In other words, if adopt cell inoculation method of the present invention, change according to the blood flow state of receptor (patient that will transplant) and cardiac valve location even contain the flox condition of the solution of cell, be seeded in autogenous cell in the vascular tissue and also can breed and cover its almost whole surface.Therefore, in general, can prepare biovalve, so that satisfy the valvular condition from adult to child, these people blood circulation amount difference in vivo, and their blood pressure is also different.

[comparative example 2]

Comparative example as routine 3-7 has carried out an experiment.Vascular tissue is stayed in the solution separately, and do not rotate up and down,, and observe the state that is inoculated into the autogenous cell in the vascular tissue according to the mode identical with example 3 so that compare with example 3.

As a result, inoculated autogenous cell, but anisotropically be distributed in the vascular tissue.Therefore, the quantity that is seeded in the autogenous cell in the vascular tissue is compared remarkable the minimizing with routine 3-7.

According to The above results, in routine 3-7 with the autogenous cell quantity of described tissue bond significantly greater than the quantity in the comparative example 2.In other words, obtained high cell inoculation effect.Specifically, when vascular tissue is immersed in the stream of beating of the solution that contains cell, improved the orientation of cell.In addition, activate cell, and improved the function of cell, consequently can inoculate the cell of bigger quantity.

The present invention can be applicable to the cells exclude process of other biological tissues, and as disclosed in the present embodiment, its blood contacts with tissue beyond the vascular tissue of containing aortic valve.In addition, the present invention can be used for the treatment of allogeneic biovalve, rather than xenograft.

In method of removing cell of the present invention, in fact not to use in the biological tissue treatment facility 10 disclosed in the present embodiment.Can use various device and device, as long as they can get rid of cell.In other words, any device can use, as long as it can provide the predetermined stream of beating of solution to be used to soak biological tissue in the cells exclude process and/or it can apply microwave to biological tissue.

As above disclosed, according to the present invention, be present in such as the germinal cell in the biological tissue of xenograft and can get rid of, and the bio-compatibility of biological tissue can improve after transplanting.

Industrial applicibility

The biovalve that the present invention can be used for taking out in the animal body that comprises the people is processed into Can be transplanted to the state in the human body.

Claims

1. biological tissue treatment facility that uses the method for removing cell of biological tissue,

Wherein, described biological tissue treatment facility comprises the holding device that is used for biological tissue is remained on cells exclude solution, and described eliminating solution can flow with the state of the stream of beating; With

Be used for the biological tissue that remains on described holding device is applied the radiation appliance of microwave.

2. biological tissue treatment facility as claimed in claim 1, wherein, the described equipment that provides can make described holding device rotate relative to radiation appliance.